DESCRIPTION (adapted from the application) The goal of the proposed studies is to begin to develop an understanding of the determinants of structural and functional heterogeneity in Detergent-Insoluble Glycosphingolipid-rich plasma membrane domains (DIGs) in the intestinal epithelia. DIGs, which include but are not limited to ultrastructurally characteristic caveolae, have been implicated in a variety of key cellular functions such as ligand-induced signal transduction, protein and lipid sorting, and vesicular trafficking. Biochemical preparations of DIGs typically consist of a mixture that include classical caveolae and non-caveolar membranes. Recent studies have suggested that classical caveolae may be biochemically or functionally distinct from the non-caveolar DIGS, and that membranes containing caveolin- I may be separated from other detergent-insoluble membranes. Heterogeneity in non-caveolar DIGs has not been formally addressed before. Our preliminary data suggest that in polarized intestinal epithelial T84 cells (which contain only rare caveolae), DIGs exhibit functional and structural heterogeneity. The observed heterogeneity has elements of epithelial cell polarity, and suggests a contribution of the cytoskeleton in establishment or expression of some of the observed differences. Proposed studies will define the patterns and determinants of heterogeneity of DIGs in polarized intestinal epithelial cells with a special emphasis on DIG/cytoskeleton interactions. These studies address the fundamental issue of cell polarity in epithelial cell biology, and may potentially identify a new aspect of intestinal epithelial cell polarity; namely, polarity in membrane microdomains. Furthermore, by using signal transduction by cholera toxin (CT) in intestinal epithelia as a classical model of DIG-mediated function, these studies extend our understanding of the pathogenesis of enterotoxicrenic secretory diarrhea.